Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) as a delivery vehicle has been well studied over the past few decades (Jensen et al., 2003; incorporated herein by reference). Originally, much of the work with this neutral, hydrophilic, biocompatible polymer focused on its use as a plasma expander; however, it has more recently been employed in the delivery of anticancer drugs (Kopecek et al., 2000; Putnam and Kopecek, 1995; Vasey et al., 1999; Duncan et al., 1992), site specific delivery in the GI tract (Yeh et al., 1995; Kopeckova et al., 1994), tumor-specific delivery of antisense oligonucleotides to their target mRNAs (Wang et al., 1999; Jensen et al., 2001), and hydrogels (Yeh et al., 1995; Wang et al., 1999; Subr et al., 1990; Rihova et al., 1997). As with other successful delivery vehicles, drug conjugates of PHPMA are well suited to pharmaceutical applications because they exhibit the enhanced permeability and retention (EPR) effect, therefore increasing the concentration of an active drug in tumor cells (i.e. 10-100 times higher concentration than that of the free drug) and decreasing the dose limiting toxicity (Matsumura and Maeda, 1986; Duncan, 1997).
Controlling the free-radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) and, therefore, controlling the molecular weight and molecular weight distribution of PHPMA is a crucial factor in the synthesis of well defined polymer-drug conjugates. The first attempts to control the polymerization of HPMA were reported by Teodorescu and Matyjaszewski (1999 and 2000) and employed atom transfer radical polymerization (ATRP) in organic media (1-butanol or ethanol) with two different ligand-initiating systems. However, these systems suffered from elevated polydispersities (Mw/Mn) and, in some cases, low conversions. An indirect method for the preparation of well-defined polymers of HPMA and PHPMA-drug conjugates was demonstrated by Godwin et al. (2001) in which N-methacryloxysuccinimide was first polymerized via ATRP followed by reaction of the succinimidyl-ester side-chains with stoichiometric amounts of 1-amino-2-propanol and an amino-terminal, peptide-linked “model drug,” glycine-glycine-β-naphthylamide.
In recent years, efforts have been focused on the controlled polymerization of water-soluble, acrylamido monomers via reversible addition-fragmentation chain transfer (RAFT) polymerization (McCormick et al., 2004; incorporated herein by reference). To date, the controlled RAFT polymerization of anionic, zwitterionic, and neutral acrylamido monomers has been reported in both organic and aqueous media employing a variety of chain transfer agents (CTA) including xanthates, dithiocarbamates, trithiocarbonates and dithioesters. All of these CTAs can afford control over the molecular weight and yield (co)polymers with low Mw/Mn values under appropriate conditions.
Most recently, attention has turned to methacrylamido monomers and the first “truly” controlled polymerization of HPMA via aqueous RAFT polymerization mediated by 4-cyanopentanoic acid dithiobenzoate (CTP) (Scales et al., 2005; incorporated herein by reference). HPMA was polymerized with good control over the molecular weight and polydispersity (Mw/Mn) in an acetic acid/sodium acetate buffer solution. The first RAFT polymerization of a cationic methacrylamido species, namely N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA), mediated by CTP in aqueous media under the same conditions used for the polymerization of HPMA has also been reported (Vasilieva et al., 2004; incorporated herein by reference). DMAPMA was also polymerized in an acetic buffer with good control over the molecular weight (Mn=47,000 g/mol, Mw/Mn=1.08), while the corresponding polymerization in water alone exhibited a loss of control due to thiocarbonylthio hydrolysis (Mn=44,500 g/mol, Mw/Mn=1.62).
The control of gene expression using specific nucleic acid sequences represents a significant step toward preventing or eliminating several genetic diseases, viruses, and cancers. Discovery of RNA interference (RNAi) has made it possible to turn off (silence) specific genes by small interfering RNA (siRNA)—19-24 base-paired RNA fragments with 2-nucleotide overhangs at the 3′ ends (Elbashir et al., 2001; McCaffrey et al., 2002; both of which are herein incorporated by reference). An increasing number of experiments using siRNA in various biological systems have overwhelmingly demonstrated siRNA's effectiveness and great potential as the next generation of RNA-based therapeutic agents. Although preliminary results suggest that siRNA is a more potent inhibitor of gene expression and is less toxic to cells than other gene silencing agents (e.g., antisense oligodeoxyribonucleic acids (ODNs) (Xu et al., 2003; Miyagishi et al., 2003; Kretschmer-Kazemi and Sczakiel, 2003; Grunweller et al., 2003; Toth et al., 2002) DNAzymes (Yokota et al., 2004; Lee et al., 2002) or ribozymes (Lee et al., 2002)), its delivery to the appropriate tissues and susceptibility to hydrolytic and enzymatic degradation in the bloodstream still pose a significant challenge (Braasch et al., 2003; Dorsett and Tuschl, 2004; Heidenreich, 2004).
One possibility for effective delivery and protection of siRNA in vivo involves stabilization with synthetic polycations or polycation containing block copolymers to form specialized interpolyelectrolyte complexes (IPECs) or block ionomer complexes (BICs), respectively. Such systems employed with other polynucleic acids are well documented and variations of this concept continue to be employed in gene delivery today (Kabanov and Kabanov, 1995; Kabanov et al., 1989; Perales et al., 1994; Wu and Wu, 1987; Izumrudov et al., 1999; Van de Wetering et al., 1999; Kabanov and Kabanov, 1998; Dautzenberg, 2001; Michaels and Miekka, 1961; Alvarez-Lorenzo et al., 2005; Andersson et al., 2004). IPEC systems used in gene therapy are composed of complexed polycations (e.g. poly(vinyl pyridine) or poly(L-lysine)) and polynucleic acids (e.g., DNA or RNA). Strong electrostatic interactions between oppositely charged polyelectrolytes (e.g., polycations and polynucleic acids) allow for “self-assembly,” which can substantially hinder or prevent enzymatic degradation of the incorporated polynucleotide in the bloodstream (Kabanov and Kabanov, 1995; Van de Wetering et al., 1999; Kabanov and Kabanov, 1998). The spontaneous formation of these complexes is largely driven by electrostatic interactions between the synthetic polycations and the “backbone” phosphate units of the polynucleotides. Furthermore, an overall gain in entropy due to the liberation of low molecular weight counterions and water during complexation increases the thermodynamic spontaneity of the process (Kabanov and Kabanov, 1998; Dautzenberg, 2001; Michaels and Miekka, 1961; Alvarez-Lorenzo et al., 2005; Andersson et al., 2004). The structural characteristics and solubility of IPECs in aqueous conditions are governed by the polymeric cation/polynucleotide phosphate (N/P) ratio and are maintained by the formation of non-stoichiometric IPECs, where the N/P ratio ≠1. These imbalanced IPECs can form two types of structures: 1) positive IPECs that contain an excess of polycations; and 2) negative IPECS that contain an excess of unoccupied or unpaired phosphates. While the preparation of these two electrosterically-stabilized IPEC systems does eliminate solubility issues observed with stoichiometric IPECs, the negative IPECS are typically not effective transfection agents and positive IPECs, due to their residual cationic nature, are often too interactive with a host of anionically charged small molecules and organelles (Kabanov and Kabanov, 1998). Unfortunately, stoichiometric IPEC systems are not very soluble in water due to their hydrophobic nature and lack of water-soluble stabilizing moieties.
More recently, the synthesis of block ionomer complexes (BICs) has provided a solution to the many solubility issues observed with conventional IPEC-based systems. Typically, BICs are formed by the complexation of a polyanion, such as DNA or RNA, with a block copolymer composed of cationic and neutral-hydrophilic block-segments. Although there are many examples of copolymers used in the formation of BICs, most incorporate poly(ethylene oxide) (PEO) as the neutral-hydrophilic block along with various cationic block types. For example, poly(ethylene oxide)-block-poly(spermine) (Kbanov and Kabanov, 1995), poly(ethylene oxide)-block-poly(L-lysine) (Katayose and Kataoka, 1996; Wolfert et al., 1996; Katayose and Kataoka, 1997), and poly(ethylene oxide)-block-poly(L-lysine-co-L-glycine) (Kabanov et al., 1996) block copolymer systems have each been used in the formation of stable BICs for gene delivery applications. The use of such neutral, hydrophilic blocks with a cationic block in lieu of a linear cationic homopolymer allows for the preparation of well-defined, electro-neutral (i.e. N/P ratio=1) complexes that are water-soluble and have a greater potential for use as effective transfection agents in gene delivery.
Because of its non-immunogenic properties and its well-documented behavior in the body, PHPMA as a neutral block in conjunction with a suitable cationic block offers great potential for improved transfection and retention behavior of the resulting BIC structures over more traditional PEO-based BIC systems. Such application of these polymers therefore renders a new generation of DNA/RNA delivery agents based on vinyl monomers prepared by a facile, controlled radical polymerization technique. Recently, it has been reported that the chemical coating of polyelectrolyte-based DNA-containing nanoparticles with multifunctional and telechelic PHPMA increased their in vivo residence times (Subr et al., 2006; incorporated herein by reference).
There is always a need for effective drug delivery agents that can be prepared through a controlled process.